The long term goal of the research program is to understand the biochemical and genetic regulation of lipid metabolism and membrane homeostasis in bacteria. The basic blueprint for bacterial membrane lipid biosynthesis is established by decades of research with Escherichia coli. However, there are numerous and significant differences in lipid metabolism between Gram-positive and Gram-negative bacteria. Gram-positive bacteria have unique enzymes for fatty acid biosynthesis, use a different activated fatty acid intermediate and acyltransferase system, and use transcriptional regulators that are distinctly different from E. coil. The medical significance of understanding this vital facet of intermediary metabolism has focused the research plan on investigating fatty acid and phospholipid synthesis in two important human pathogens: Staphylococcus aureus and Streptococcus pneumoniae. These organisms represent two major branches of lipid metabolic diversity in Gram-positive bacteria. Because type II fatty acid synthesis is a vital facet of bacterial physiology, there is considerable interest in the therapeutic potential of pathway inhibitors. However, Gram-positive bacteria can utilize extracellular fatty acids and may circumvent pathway inhibitors by the acquisition of host-derived fatty acids. One aim is to identify and characterize the unknown enzymes in the pathway for the incorporation of exogenous fatty acids in Gram-positive bacteria. A detailed biochemical understanding of this process will allow us to determine if exogenous fatty acids can overcome fatty acid synthesis inhibitors. The second aim is to determine the mechanism for skin antibacterial defense by fatty acid intoxication. Fatty acids have been known for decades to be a component of the innate defense system and toxic to Gram-positive bacteria. We propose a completely new mechanism of action for skin fatty acids. Fatty acid intoxication arises from the inability of specific structures to be utilized by the enzymes of lipid metabolism resulting in the accumulation of intracellular fatty acids and the collapse of the membrane proton gradient. A mechanistic understanding of this important host defense mechanism will provide a rational basis for designing novel fatty acid structures with increased efficacy. The final goal is to identify the regulatory factors and target proteins responsible for the tight biochemical control of membrane lipid biosynthesis in Gram-positive pathogens. Fatty acid synthesis is stringently regulated at the biochemical level to ensure a balanced membrane lipid composition. A system- oriented metabolomics approach will be developed to study pathway regulation. The analysis of pathway intermediates following physiological perturbations will identify the candidate regulatory ligands and target enzymes that will be biochemically characterized. The systematic analytical workflow we will develop can be applied to study any bacterium, and the outcome of these studies in pathogens will suggest a unique approach to developing new therapeutics. Structural mimics of the key regulatory ligand(s) could be developed to block membrane lipid biosynthesis.